During the past several years we have developed a novel expression system for gap junction channels, using communication deficient mammalian cells to generate cell lines in which cDNAs encoding gap junction proteins (connexins) are stably expressed. This approach differs from the more commonly used system, injection of connexin mRNA into Xenopus oocytes, in several ways that facilitate the study of the expressed gap junction channels, one of the most notable being that dual whole cell recording from pairs of mammalian cells permits examination of single channel properties. Stable transfectants currently available include a number of mammalian gap junction proteins, as well as that of an amphibian that show marked voltage sensitivity. Moreover, we have expressed several mutant constructs designed to test various hypotheses concerning gating domains of the channel protein. This grant application seeks to use these various cell lines to answer several questions that have been important to the gap junction field but have been difficult to address using primary cell cultures. We intend to 1) Determine the effect of phosphorylation state of the gap junction proteins on physiological properties of the channels that they form, 2) determine the effects of charge substitutions within the third membrane spanning domain on gap junction charge and size selectivity, 3) identify the portions of the gap junction protein that form the regions sensitive to pH and to transjunctional voltage, and 4) examine the properties of paired connexin molecules of different types. These studies should enormously "tend our understanding of the properties of gap junction channels and may result in novel molecular approaches to increase or decrease gap junction function in normal and pathological tissues.